Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.

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Abstract

C4b-binding protein (C4BP), an important inhibitor of complement activation, has a unique spider-like shape. It is composed of six to seven identical alpha-chains with or without a single beta-chain, the chains being linked by disulfide bridges in their C-terminal parts. To elucidate the structural requirements for the assembly of the alpha-chains, recombinant C4BP was expressed in HEK 293 cells. The expressed C4BP was found to contain six disulfide-linked alpha-chains. Pulse-chase analysis demonstrated that the recombinant C4BP was rapidly synthesized in the cells and the polymerized C4BP appeared in the medium after 40 min. The alpha-chains were polymerized in the endoplasmic reticulum (ER) already after 5 min chase. The polymerization process was unaffected by blockage of the transport from the ER to the Golgi mediated by brefeldin A or low temperature (10 degrees C). The C-terminal part of the alpha-chain (57 amino acids), containing 2 cysteine residues and an amphiphatic alpha-helix region, was required for the polymerization. We constructed and expressed several mutants of C4BP that lacked the cysteine residues and/or were truncated at various positions in the C-terminal region. Gel filtration analysis of these variants demonstrated the whole alpha-helix region to be required for the formation of stable polymers of C4BP, which were further stabilized by the formation of disulfide bonds.

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Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology

Keywords

  • Hydrolysis, Protein Conformation, Receptors, Complement : chemistry, Complement : genetics, Complement : isolation & purification, Recombinant Proteins : chemistry, Recombinant Proteins : genetics, Recombinant Proteins : isolation & purification, Spectroscopy, Fourier Transform Infrared, Human, DNA Primers, Chymotrypsin : metabolism, Cell Line, Biopolymers : chemistry, Base Sequence
Original languageEnglish
Pages (from-to)9349-9357
JournalBiochemistry
Volume41
Issue number30
Publication statusPublished - 2002
Publication categoryResearch
Peer-reviewedYes